Muscular dystrophy chimeric cells and method for treating muscular dystrophies

ABSTRACT

A Muscular Dystrophy Chimeric Cell generated by ex vivo fusion of a myoblast with a second myoblast, mesenchymal stem cell, or stromal cell is described as is the use of the same in the treatment of a muscular dystrophy.

INTRODUCTION

This application is a continuation of U.S. Ser. No. 15/580,783 filedDec. 8, 2017, which is the National Phase application ofPCT/US2016/036821 filed Jun. 10, 2016, which claims benefit of priorityto U.S. Provisional Patent Application Ser. No. 62/174,122, filed Jun.11, 2015, the contents of which are incorporated herein by reference inits entirety.

BACKGROUND

Muscular dystrophies (MD) are a group of more than 30 genetic diseasescharacterized by progressive weakness and degeneration of the skeletalmuscles that control movement. Some forms of MD are seen in infancy orchildhood, while others may not appear until middle age or later. Thedisorders differ in terms of the distribution and extent of muscleweakness (some forms of MD also affect cardiac muscle), age of onset,rate of progression, and pattern of inheritance.

Duchenne muscular dystrophy (DMD), the most common, lethal X-chromosomelinked progressive muscle-wasting disorder, is caused by dystrophin genemutations resulting in the absence of dystrophin, a protein involved inmaintaining the integrity of muscle. DMD affects 1 in every 3500 malebirths. Onset is between 3 and 5 years and the disorder progressesrapidly. Most boys are unable to walk by age 12, and later need arespirator to breathe. Girls in these families have a 50 percent chanceof inheriting and passing the defective gene to their children.Debilitated patients cannot partake in routine activities; most arewheelchair-dependent by the age of 12. Life expectancy of DMD patientsis 25. Boys with Becker MD (very similar to but less severe thanDuchenne MD) have faulty or not enough dystrophin.

Facioscapulohumeral MD usually begins in the teenage years. It causesprogressive weakness in muscles of the face, arms, legs, and around theshoulders and chest. It progresses slowly and can vary in symptoms frommild to disabling.

Myotonic MD is the disorder's most common adult form and is typified byprolonged muscle spasms, cataracts, cardiac abnormalities, and endocrinedisturbances. Individuals with myotonic MD have long, thin faces,drooping eyelids, and a swan-like neck.

Muscular dystrophies are caused by progressive degeneration of skeletalmuscle fibers. Lack of one of several proteins located either at theplasma membrane or, less frequently, within internal membranes increasesthe probability of damage during contraction, and eventually leads tofiber degeneration, accompanied by severe local inflammation withinfiltration of immune-competent cells. In the most severe forms, suchas Duchenne Muscular Dystrophy, regeneration is exhausted and skeletalmuscle is progressively replaced by fat and fibrous tissue. Thiscondition leads the patient to progressive weakness and eventually deathby respiratory and/or cardiac failure.

At present, an effective therapy for MD has not been found yet, andimportance is placed on rehabilitation for retarding the progression ofsymptoms or respiratory management using mechanical ventilators or thelike. Drug therapy includes corticosteroids (steroids), but they havestrong side effects and have not produced sufficient therapeutic effect.Experimental therapies such as regenerative therapy (transplantation ofstem cells and myoblasts) (Meregalli, et al. (2010) BioDrugs 24:237-247;U.S. Pat. Nos. 7,341,719; 7,887,793; and 7,452,529), gene therapy(functional dystrophin gene transfer, antisense morpholino-mediatedskipping of mutated exons), alternative drug therapy (read-through ofnonsense mutations) and the like have been suggested. However, there isan urgent need to develop new, more effective strategies to treatpatients with MD.

SUMMARY OF THE INVENTION

This invention provides a Muscular Dystrophy Chimeric Cell (MDCC)prepared by fusion of (a) a first myoblast; and (b) a second myoblast,mesenchymal stem cell, or stromal cell, wherein at least one of thefirst myoblast, second myoblast, mesenchymal stem cell, or stromal cellis from a healthy donor. In some embodiments, the first myoblast andsecond myoblast are from different donors. In other embodiments, thefirst myoblast, second myoblast, mesenchymal stem cell, or stromal cellis autologous or allogeneic. In particular embodiments, the healthydonor is the subject's father. In further embodiments, the mesenchymalstem cells are derived from bone marrow or adipose tissue and the MDCCsecretes one or more immunomodulatory cytokines and growth factors,e.g., insulin-like growth factor 1, hepatocyte growth factor andmyostatin. A composition containing the MDCC, a kit, and a method ofadministering the MDCC by intravenous injection, intra-bone injection orintramuscular injection in the treatment of a muscular dystrophy such asDuchenne Muscular Dystrophy are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the ex vivo fusion procedure to create human MDCC. In oneembodiment, human myoblasts are obtained from a DMD patient's muscle andare fused with myoblasts or mesenchymal stem cells (MSC) from a healthydonor. Prior to fusion, cells are fluorescently labeled with PKH26 orPKH67, respectively. Cell fusion of fluorescently labeled cells isperformed using polyethylene glycol (PEG). Double (PKH26 and PKH67)stained cells that undergo fusion are selected via fluorescentlyactivated cells sorting (FACS; BD Asterios). These cells are deliveredthrough intramuscular injection into DMD patient deteriorating muscles.

FIG. 2 shows flow cytometry analysis of ex vivo created (by fusion)human MD chimeric cells (hMDCC). Shown are dot plots of unstainedmyoblasts, PKH26-stained myoblasts, PKH67-stained mesenchymal stem cells(MSC), and fused PKH26/PKH67-stained cells, confirming creating ofhMDCC.

FIGS. 3A and 3B show the results of an in vivo muscular contractilitytest. Muscle force (FIG. 3A) and percent fatigue (FIG. 3B) measurementswere normalized with the muscle weights. hMDCC-treated animals showedimproved muscle force (p=0.04) and fatigue tolerance 90 days after MDCClocal injections.

FIGS. 4A and 4B show the results of an ex vivo muscular contractilitytest. mMDCC-treated gastrocnemius muscles showed increased contractilestrength expressed after the maximal sine wave (p=0.039; FIG. 4A) andmaximal percent strain (FIG. 4B), compared to control, untreated muscle.

FIGS. 5A and 5B show the results of in vivo muscular contractility test.Muscle force (FIG. 5A) and Percent fatigue (FIG. 5B) measurements werenormalized with the muscle weights. mMDCC-treated animals showedimproved muscle force and fatigue tolerance (p=0.05) 30 days after MDCClocal injections.

DETAILED DESCRIPTION OF THE INVENTION

MDCC compositions and methods for treating muscular dystrophies inpatients have now been developed. MDCC lines are created by ex vivofusion of a first population of myoblasts (autologous or allogeneic)with a second population of myoblasts, mesenchymal stem cells or stromalcells. Administration of the MDCC enables simultaneous delivery ofmyogenic and mesenchymal origin cells to patients. Accordingly, thisinvention provides MDCCs prepared by fusion of myoblasts from a subjectsuffering from a muscular dystrophy and myoblasts from a healthy donor;myoblasts from a subject suffering from a muscular dystrophy andmesenchymal stem cells; myoblasts from a healthy donor and mesenchymalstem cells; myoblasts from a subject suffering from a muscular dystrophyand stromal cells; or myoblasts from a healthy donor and stromal cells.In particular embodiments, the MDCCs of this invention find use in amethod for treating muscular dystrophies.

This invention differs from other cell-based therapies since thecombined therapy introduces myoblasts differentiating into myocytes,whereas MSC are known for reduced alloreactivity, plasticity andpotential for de-differentiation into myoblasts in damaged tissue.Combination of myoblasts/MSC characteristics, including the secretion ofimmunomodulatory cytokines and growth factors, supports MDCCengraftment, tolerance and regeneration of muscle under favorablemicroenvironment conditions. On the other hand, myoblast/myoblast MDCCcharacteristics and capacity to spontaneously fuse offer the ability toeither engraft and resupply the muscle stem cell niche or fuse with therecipient myoblast after treatment, providing better outcomes comparedto myoblasts/MSC MDCC. Cells harvested from DMD patient may be fusedwith healthy, dystrophin-positive stem cells from haploidentical malerelative, i.e., father or from haplo-matched donor in cell banks. Inthis manner, the MDCC shares surface antigens of self andhaplo-identical origin, reducing the risk of rejection of transplantedMDCC. Further, given that the MDCCs are not genetically modified (i.e.,by recombinant methods) and do not require immune suppression, use ofthe cells of this invention provides a safe alternative to conventionaltherapies.

For the purposes of this invention, a “chimeric cell” or “hybrid cell”is a cell that is constructed from a somatic cell hybridization (or awhole cell hybridization) of, for example, two or more biological cells(parent cells). The parent or donor cells can be obtained from eitherthe same donor or cell lineage or different donors or cell lineage.While the MDCC of this invention is referred to as “a chimeric cell,”said chimeric cell is intended to mean a single cell or a population ofcells.

As used herein, a donor is a subject who provides a cell used in thepreparation of a chimeric cell of this invention. The donor can be asubject with a muscular dystrophy or a healthy donor, i.e., anindividual not suffering from the same genetic disorder. The donor canbe the genetic father (parent) of a subject (son) with a musculardystrophy or a cell bank donor. In particular embodiments, at least oneof the donors is a healthy subject. In certain embodiments, the healthydonor is the subject's father. In other embodiments, the first myoblast,a second myoblast, mesenchymal stem cell or stromal cell is autologousor allogeneic. Further, the donor may be any mammal including a human,mouse, rat, dog, cat, horse, and the like. In particular embodiments,the donor is human.

As is convention in the art, a myoblast refers to a primitive musclecell having the potential to develop into a muscle fiber. Myoblasts arecharacterized by expression of desmin and CD56, and can be obtained fromfetal or adult tissue using a method known in the art. See, e.g., WO93/03768, which discloses the isolation of myoblasts from a crude cellpopulation by flow cytometry (e.g., FACs). Alternatively, a myoblast canbe obtained by growing and propagating muscle biopsy-derived myoblastsin culture. See, e.g., Springer, et al. (1997) In: Current HumanGenetics. Unit 13.4, Boyle Ed. John Wiley & Sons, NY. In accordance withsome embodiments of this invention, a myoblast from a healthy donor isfused with a myoblast from a subject with a muscular dystrophy. Inparticular embodiments, a first myoblast and a myoblast are fromdifferent donors.

“Mesenchymal stem cells” (also referred to as “MSCs”) can give rise toconnective tissue, bone, cartilage, and cells in the circulatory andlymphatic systems. Mesenchymal stem cells are found in the mesenchyme,the part of the embryonic mesoderm that consists of loosely packed,fusiform or stellate unspecialized cells. Mesenchymal stem cells can beobtained by conventional methods and can be identified one or more ofthe following markers: CD29, CD31⁻, CD34⁻, CD44 CD45⁻, CD51, CD73,CD90/Thy-1, CD105, CD166, Integrin α1, PDGF Rα, Nestin, Sca-1⁺, SCFR/c-Kit, STRO-1, and VCAM-1. In some embodiments, the mesenchymal stemcells are derived or obtained from bone marrow (BM) or adipose tissue(ASC). In particular embodiments, the mesenchymal stem cells are derivedor obtained from human bone marrow.

The term “stromal cell” or “adherent stromal cell” is intended to mean acell defined by its ability to adhere and proliferate in tissue-culturetreated petri dishes with or without other cells and/or elements foundin loose connective tissue, including but not limited to, endothelialcells, pericytes, macrophages, monocytes, plasma cells, mast cells andadipocytes. Any suitable method can be used to obtain stromal cells.See, e.g., Tondreau, et al. (2005) Stem Cells 23:1105-1112. Inparticular embodiments, the stromal cell is derived or obtained frombone marrow (BM) or cord blood (CB). In other embodiments, the stromalcell is a CD133⁺ stromal cell.

The cells used in the preparation of the MDCCs of this invention can beisolated and optionally purified. As used herein the term “isolated” ismeant to describe a cell of interest that is in an environment differentfrom that in which the element naturally occurs. “Purified” as usedherein refers to a cell removed from an environment in which it wasproduced and is at least 60% free, preferably 75% free, and mostpreferably 90% free from other components with which it is naturallyassociated or with which it was otherwise associated with duringproduction.

Purification and/or identification of cells of interest can be achievedthrough any means known in the art, for example immunologically.Histochemical staining; flow cytometry, fluorescence activated cellsorting (FACS), western blot analysis, enzyme-linked immunosorbent assay(ELISA), etc. may be used. Flow immunocytochemistry may be used todetect cell-surface markers, immunohistochemistry (for example, of fixedcells) may be used for intracellular or cell-surface markers. Westernblot analysis may be conducted on cellular extracts. Enzyme-linkedimmunosorbent assay may be used for cellular extracts or productssecreted into the medium. Antibodies for the identification of stem cellmarkers may be obtained from commercial sources, for example fromChemicon International, (Temecula, Calif.).

In some embodiments, a donor cell used in the preparation of the MDCC ofthis invention is autologous or heterologous to the subject beingtreated. In other embodiments, a donor cell used in the preparation ofthe MDCC of this invention is allogeneic to said subject. In certainembodiments, the donor cells are HLA (human leukocyte antigen)-matched.Representative sources of donor cells used for the preparation of theMDCCs of the invention are listed in Table 1.

TABLE 1 Father Son MSC Myoblast Myoblast Myoblast Bone Marrow (BM)Myoblast MDSC Myoblast CD133⁺ (BM) Myoblast MSC Muscle-derived stem cell(MDSC) Myoblast MDSC Bone Marrow MDSC MDSC MDSC CD133⁺ (BM) MDSCMyoblast MSC Myoblast Bone Marrow ASC Myoblast ASC ASC Father Father MSCMyoblast CD133⁺ (BM) Myoblast Bone Marrow Myoblast MSC MDSC CD133⁺ (BM)MDSC Bone Marrow MDSC ASC Myoblast Cell Bank Son CD133⁺ (BM) MyoblastCD133⁺ (CB) Myoblast MSC Myoblast CD133⁺ (BM) MDSC CD133⁺ (CB) MDSC MSCMDSC Cell Bank Father CD133⁺ (BM) Myoblast CD133⁺ (CB) Myoblast MSCMyoblast CD133⁺ (BM) MDSC CD133⁺ (CB) MDSC MSC MDSC

In particular embodiments, the MDCC is produced by fusing:

a) a human myoblast from a subject suffering from a muscular dystrophyand a human myoblast from a healthy donor;

b) a human myoblast from a subject suffering from a muscular dystrophyand a human mesenchymal stem cell from a healthy donor;

c) a human myoblast from a healthy donor and a human mesenchymal stemcell from a healthy donor;

d) a human myoblast from a subject suffering from a muscular dystrophyand a human stromal cell from a healthy donor; or

e) a human myoblast from a healthy donor and a human stromal cell from ahealthy donor.

The MDCC of this invention is prepared by ex vivo fusion of twodifferent donor cells. By “ex vivo” it is meant that cells aremanipulated outside of the body. Cell fusion is a process in which twoor more cells merge into one by fusing their plasma membranes. MDCCs canbe prepared by cell fusion methods known in the art, including, but notlimited to, exposure of cells to fusion-promoting chemicals, such aspolyethylene glycol (PEG); the use of inactivated virus, such as Sendaivirus; and the use of electrical stimulation. See, e.g., Kennett (1979)Methods Enzymol. 58:345-359 for a review of the commonly used methodsbased upon Sendai virus induced cell fusion, or cell fusion induced bypolyethylene glycol (PEG). Briefly, cells to be fused are incubated witha fusogenic agent, such as Sendai virus or PEG. Centrifugation oragitation may be used to encourage clumping and close apposition of thecell membranes. Variables such as time, temperature, cell concentrationand fusogenic agent concentration may be optimized for each cellcombination. With respect to electro fusion, short electric pulses arepassed through mixtures of cells to stimulate fusion. See, e.g., Neil &Zimmermann (1993) Methods Enzymol. 220:174-196.

In certain embodiments, the MDCCs are prepared by polyethylene glycolcell fusion. After fusion, cell lines representing eithermyogenic/myogenic or myogenic/MSC origin are separated, cultured andcharacterized to confirm the myoblast and MSC specific markers and HLAclass I types of cell donor origin.

Prior to fusion, the donor cells may or may not be cultured to increasetheir number. Further, the donor cells may or may not be labeled (e.g.,with a membrane dye) to monitor fusion of the donor cells. By way ofillustration, myoblasts from a subject suffering from a musculardystrophy are labeled with PKH26-red and myoblasts from a healthy donorare labeled with PKH67-green.

In some embodiments, the MDCC of this invention secretes one or moreimmunomodulatory cytokines and growth factors. In certain embodiments,the immunomodulatory cytokines and growth factors include insulin-likegrowth factor 1 (IGF-1), hepatocyte growth factor (HGF) and myostatin.In further embodiments, the MDCC of this invention produces dystrophin.

The MDCC of this invention is of particular use in the treatment ofmuscular dystrophies. Accordingly, this invention also provides methodsof treating muscular dystrophies in a subject in need thereof byadministering to the subject the MDCC of the invention or a compositioncontaining the MDCC in an amount effective to treat the dystrophies.“Treating” a subject having a disease or disorder means accomplishingone or more of the following: (a) reducing the severity of the disease;(b) arresting the development of the disease or disorder; (e) inhibitingworsening of the disease or disorder; (d) limiting or preventingrecurrence of the disease or disorder in patients that have previouslyhad the disease or disorder; (e) causing regression of the disease ordisorder; (f) improving or eliminating the symptoms of the disease ordisorder; and (g) improving survival.

As indicated herein, muscular dystrophies are a group of geneticdiseases characterized by progressive weakness and degeneration of theskeletal muscles that control movement. Examples of muscular dystrophiesinclude Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, LimbGirdle Muscular Dystrophy, Myotonic Muscular Dystrophy,Facioscapulohumeral Muscular Dystrophy, Oculopharyngeal musculardystrophy, Emery-Dreifuss muscular dystrophy, Fukuyama-type congenitalmuscular dystrophy, Miyoshi myopathy, Ullrich congenital musculardystrophy, Steinert Muscular Dystrophy. In certain embodiments, themuscular dystrophy is Duchenne muscular dystrophy (DMD).

In accordance with the method of treatment, a MDCC or compositioncontaining the same is administered to a subject having a musculardystrophy. In some embodiments, a combination of MDCCs of this inventioncan be administered. The MDCC or combination of cells can beadministered by engraftment, wherein the cells are injected into thesubject, for example, intravenously, intra-muscularly, intra-arterially,intra-bone and the like. In certain embodiments, administration involvesengrafting about 10², 10⁴, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹², or morecells. The number of cells engrafted may be chosen based on the route ofadministration and/or the severity of the condition for which the cellsare being engrafted. Advantageously, the MDCC of this invention willsuccessfully engraft and complement the function of defected muscles ofmuscular dystrophy patients.

Compositions containing the MDCC or combinations of MDCCs can beprepared by combining the cell or combination of cells with apharmaceutically acceptable carrier or aqueous medium. The phrase“pharmaceutically or pharmacologically acceptable” refers to molecularentities and compositions that do not produce adverse, allergic, orother untoward reactions when administered to an animal or a human. Asused herein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with thecells of the present disclosure, its use in therapeutic compositions iscontemplated. Pharmaceutical compositions can be determined by oneskilled in the art depending upon, for example, the intended route ofadministration, delivery format and desired dosage. See, for example,REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (A. R. Gennaro, ed.),1990, Mack Publishing Company.

The compositions of the invention can be incorporated in an injectableformulation. The formulation may also include the necessaryphysiologically acceptable carrier material, excipient, lubricant,buffer, surfactant, antibacterial, bulking agent (such as mannitol),antioxidants (ascorbic acid or sodium bisulfite) and the like.

Acceptable formulation materials preferably are nontoxic to recipientsat the dosages and concentrations employed. The pharmaceuticalcomposition may contain formulation materials for modifying, maintainingor preserving, for example, the pH, osmolarity, viscosity, clarity,color, isotonicity, odor, sterility, stability, rate of dissolution orrelease, adsorption or penetration of the composition. Suitableformulation materials may include, but are not limited to, amino acids(such as glycine, glutamine, asparagine, arginine or lysine);antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite orsodium hydrogen-sulfite); buffers (such as borate, bicarbonate,Tris-HCl, citrates, phosphates or other organic acids); bulking agents(such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA; complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides,disaccharides, and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as PLURONICS, PEG, sorbitan esters, polysorbates such aspolysorbate 20 and polysorbate 80, TRITON, trimethamine, lecithin,cholesterol, or tyloxapal); stability enhancing agents (such as sucroseor sorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol, or sorbitol);delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, Id.

The primary vehicle or carrier in a pharmaceutical composition may beeither aqueous or nonaqueous in nature. For example, a suitable vehicleor carrier may be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplaryvehicles. Pharmaceutical compositions can comprise Tris buffer of aboutpH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may furtherinclude sorbitol or a suitable substitute therefore. Pharmaceuticalcompositions of the invention may be prepared for storage by mixing theselected composition having the desired degree of purity with optionalformulation agents (REMINGTON'S PHARMACEUTICAL SCIENCES, Id.) in theform of a lyophilized cake or an aqueous solution.

The cell or composition can be provided by sustained release systems, byencapsulation or by implantation devices. The compositions may beadministered by bolus injection or continuously by infusion, or byimplantation device. The composition also can be administered locallyvia implantation of a membrane, sponge or another appropriate materialonto which the cell or cells have been absorbed or encapsulated. Wherean implantation device is used, the device may be implanted into anysuitable tissue or organ. The injections may be given as a one-timetreatment, repeated (daily, weekly, monthly, annually etc.) in order toachieve the desired therapeutic effect.

Cell encapsulation methodology has been previously described whichallows transplantation of encapsulated cells in treatment of Parkinson'sdisease (Tresco, et al. (1992) ASAIO J. 38:17-23) or Amyotrophic lateralsclerosis (Aebischer, et al. (1996) Hum. Gene Ther. 7:851-860). Inaccordance with this embodiment, cells are encapsulated by compoundswhich form a microporous membrane. Capsules, for example approximately 1cm in length, containing the cells of interest may be prepared employinga hollow microporous membrane fabricated from poly-ether-sulfone (PES)(Akzo Nobel Faser AG, Wuppertal, Germany; Déglon, et al. (1996) Hum.Gene Ther. 7:2135-2146).

The compositions of the invention can be delivered parenterally. Whenparenteral administration is contemplated, the therapeutic compositionsfor use in this invention may be in the form of a pyrogen-free,parenterally acceptable aqueous solution. A particularly suitablevehicle for parenteral injection is sterile distilled water. Preparationcan involve the formulation with an agent, such as injectablemicrospheres, bio-erodible particles, polymeric compounds (such aspolylactic acid or polyglycolic acid), beads or liposomes, that mayprovide controlled or sustained release of the cell or cells, which maythen be delivered via a depot injection. Formulation with hyaluronicacid has the effect of promoting sustained duration in the circulation.Implantable drug delivery devices may be used to introduce the desiredcomposition.

These compositions may also contain adjuvants such as preservative,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms can be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid and the like. It may also be desirableto include isotonic agents such as sugars, sodium chloride and the like.

Supplementary active ingredients also can be incorporated into thecompositions. The active compositions of the present disclosure mayinclude classic pharmaceutical preparations. Administration of thesecompositions according to the present disclosure will be via any commonroute so long as the target tissue is available via that route. Suchroutes include oral, nasal, buccal, rectal, vaginal or topical route.Alternatively, administration may be by orthotopic, intradermal,subcutaneous, intraperitoneal, or intravenous injection. Intramuscularinjection will be preferred. Such compositions would normally beadministered as pharmaceutically acceptable compositions.

As used herein, the term “amount effective,” “effective amount” or a“therapeutically effective amount” refers to an amount of the cell orcomposition of the invention sufficient to achieve the desired result.The amount of the cell or composition which constitutes an “effectiveamount” or “therapeutically effective amount” may vary depending on theseverity of the disease, the condition, weight, or age of the patient tobe treated, the frequency of dosing, or the route of administration, butcan be determined routinely by one of ordinary skill in the art. Aclinician may titer the dosage or route of administration to obtain theoptimal therapeutic effect.

The present invention is also directed to a kit to for the treatment ofa muscular dystrophy. The kit is useful for practicing the inventivemethod of treating a muscular dystrophy. The kit is an assemblage ofmaterials or components, including at least one of the inventivecompositions. Thus, in some embodiments the kit a fusogenic agent forcarrying out ex vivo cell fusions, and one or more donor cells (e.g.,donor cells from a cell bank), and optionally materials for obtainingdonor cells, as described above.

The exact nature of the components configured in the inventive kitdepends on its intended purpose. For example, some embodiments areconfigured for the purpose of treating a muscular dystrophy. In oneembodiment, the kit is configured particularly for the purpose oftreating human subjects. In another embodiment, the kit is configuredparticularly for the purpose of treating adult, human subjects. Inanother embodiment, the kit is configured particularly for the purposeof treating children. In another embodiment, the kit is configuredparticularly for the purpose of treating DMD. In another embodiment, thekit is configured particularly for the purpose of treating BMD. Inanother embodiment, the kit is configured particularly for the purposeof providing continuous daily use dosages. In another embodiment, thekit is configured particularly for the purpose of providing as neededuse dosages. In further embodiments, the kit is configured forveterinary applications, treating subjects such as, but not limited to,farm animals, domestic animals, and laboratory animals.

Instructions for use may be included in the kit. “Instructions for use”typically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome,such as to treat muscular dystrophy, to treat BMD, or to treat DMD.Optionally, the kit also contains other useful components, such as,diluents, buffers, pharmaceutically acceptable carriers, syringes,catheters, applicators, pipetting or measuring tools, bandagingmaterials or other useful paraphernalia as will be readily recognized bythose of skill in the art.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures. The components are typicallycontained in suitable packaging material(s). As employed herein, thephrase “packaging material” refers to one or more physical structuresused to house the contents of the kit. The packaging material isconstructed by well-known methods, preferably to provide a sterile,contaminant-free environment. The packaging materials employed in thekit are those customarily utilized in therapeutic treatment. As usedherein, the term “package” refers to a suitable solid matrix or materialsuch as glass, plastic, paper, foil, and the like, capable of holdingthe individual kit components. The packaging material generally has anexternal label which indicates the contents and/or purpose of the kitand/or its components.

The following non-limiting examples are provided to further illustratethe present invention.

Example 1: Ex Vivo Preparation of Human Muscular Dystrophy ChimericCells (hMDCC)

Ex vivo fusions of allogenic human myoblasts and MSC or myoblasts fromtwo unrelated donors were performed, using polyethylene glycol technique(FIG. 1). Briefly, commercially available (Lonza, Inc.) human myoblastsand MSC were separately cultured for 6 to 10 days. Next, cells werefluorescently labeled using either PKH-26 (red) or PKH-67 (green)tracking dye, as shown in FIG. 1. Fusion was performed using PEG. Cellspresenting double (PKH26 and PKH67) staining were selected viafluorescently activated cell sorting (FACS; BD ASTERIOS). To confirmfusion, double (PKH26 and PKH67) labeled MDCC were evaluated usingconfocal microscopy and flow cytometry (FIG. 2). Morphology of MDCC wasassessed using transmission electron microscopy. The MDCC were confirmedby the presence of two nuclei, fused cell membrane and fused cytoplasm.

Flow cytometry was used to assess phenotype changes of MDCC at 7, 14, 21and 30 days following fusion. MDCC were tested for the expression ofmuscle-specific markers (Anti-Myogen in, Anti-hMyosin Heavy Chain,Anti-mMYF-5) and MSC markers (CD105, CD73, CD90). The results are shownin Table 2. Notably, the MDCC did not express CD45 or CD8 markers, whichare characteristic for hematopoietic cells.

TABLE 2 Sample Myoblast Myoblast MDCC after Phenotype Donor A Donor BFusion A + B Anti-myogenin + − + Anti-hMyosin Heavy Chain + − +Anti-mMYF-5 + − + CD105 − + + CD73 − + + CD90 − + +

Additionally, MDCC were analyzed by FISH to detect sex chromosomes andviability staining (Trypan blue). Further, MDCC were cultured for 30days to test proliferation and secretory properties via ELISA assay.Moreover, using immunofluorescence staining, the expression ofdystrophin was demonstrated in the human MDCC. In addition, in vitroresults confirmed myogenic differentiation potential of hMDCC. Afterfusion, hMDCC were placed in specific myogenic differentiation medium(low serum medium supplemented with insulin, PROMOCELL) for 7 days.Skeletal myosin heavy chain expression, a marker of skeletal myocytedifferentiation, was observed in all hMDCC lines.

To assess genotype and confirm cell fusion, Polymerize ChainReaction-Reverse Sequence-Specific Probe (PCR-rSSOP) and short-tandemrepeat-PCR (STR-PCR) was performed on MDCC, detecting HLA class I and IIand specific gene combination from both donors (Tables 3 and 4,respectively). This analysis indicated that MDCC presented HLA allelesderived from both donor cells and showed the presence of genetic markersspecific for both fusion donor cells.

TABLE 3 Sample Myoblast Myoblast MDCC after Fusion HLA Type Donor ADonor B (A + B) A 01, 31 30, 30 01, 31, 30 B 08, 39 42, 57 08, 39, 42,57 Cw 07, 07 17, 18 07, 07, 17, 18 Bw 6 6, 4 6, 4 DRB1 03 (17), 14 08,03 (18) 03 (17), 14, 08, 03 (18) DQB1 02, 03 (7) 03 (7), 04 02, 03 (7),03 (7), 04 DR52, DR53 52 52 52

TABLE 4 Sample Genetic Myoblast Myoblast MDCC after Marker Donor A DonorB Fusion (A + B) D5S818 11, 12 11, 12 11, 12 D7S820 8, 10 10 8, 10 Th018, 9, 3 6, 7 8, 9, 3, 6, 7 AMEL X, Y X X, Y TPOX 9, 11 8, 9 9, 11, 8CSF1PO 11 12 11, 12 D21S11 28, 30 30, 31 28, 30, 31

In vitro culturing results showed proliferative potential, long-termviability and differentiation of DMDCC to the myocyte lineage. Theexpression of dystrophin was maintained by DMDCC up to 30 dayspost-fusion. Secretion of cytokines by DMDCC was confirmed by ELISAassay.

In in vivo studies, engraftment of locally administered hMDCC inmdx/scid mouse model was assessed. Five groups of mice were tested(Table 5). The first two groups included MDCC of myoblast/myoblast orMSC/myoblast origin (dose 0.5×10⁶) delivered through multipleintramuscular injections following a standardized template, to the leftgastrocnemius muscle of mdx/scid mice. Control groups included treatmentwith vehicle, treatment with unfused myoblasts (dose 0.5×10⁶), ortreatment with mixed MSCs and myoblasts (dose 0.5×10⁶) via intramuscularinjection. Outcomes measured included in vivo muscle function,dystrophin expression in treated muscles as well as MDCC engraftment at1 week and 12 week time-points.

Dystrophin expression was used as a specific marker for hMDCC since ithad been confirmed that both hMDCC lines expressed dystrophin. After 7days following local intramuscular delivery of hMDCC, successfulengraftment of hMDCC was shown. In addition, locally increaseddystrophin expression (12%) was observed as early as 7 daypost-transplant as compared with the lack of dystrophin expression inmdx/scid controls. Furthermore, at 90 days 17% of dystropin expressionwas observed.

TABLE 5 Time- point No. of Treatment (days) Animals OutcomesMyoblast/MSC MDCC 7 3 MDCC engraftment and dystrophin expression (DE) 903 Muscle function and structure, DE Myoblast/Myoblast 7 3 MDCCengraftment and MDCC DE 90 3 Muscle function and structure, DE Myoblastswithout 7 3 MDCC engraftment and fusion DE 90 3 Muscle function andstructure, DE Myoblast and MSC 7 3 MDCC engraftment and without fusionDE 90 3 Muscle function and structure, DE Vehicle 7 3 MDCC engraftmentand DE 90 3 Muscle function and structure, DE

Another set of experimental groups (vehicle and hMDCC treated mdx/scidand wild type snj mice, n=3) were tested in motor function tests,including grip strength measurements and a wire hanging test. Ninetydays after hMDCC therapy delivery, mice receiving hMDCC showedimprovement (p=0.037) of grip strength and tolerance to fatigue on wire(mdx/scid mice, 50 gF; hMDCC therapy, 85-90 gF). Functional improvementwas observed in groups treated with both MDCC lines when compared tovehicle treated groups until day 42. After this time-point onlymyoblast/myoblast hMDCC maintained increased muscle strength throughoutthe 90 day follow-up period. By comparison, grip strength valuesreturned to baseline levels after 42 days in mice treated withMSC/myoblast hMDCC. The unfused cell treatment control groups showedtemporary motor function improvement in the first 21 days only.

Gastrocnemius muscles harvested at 90 days after hMDCC delivery wereanalyzed in an ex vivo contractility test to assess muscle strengthevoked by electric stimulation. Results showed that myoblast/myoblasthMDCC treated (FIG. 3A) muscle had significantly stronger contraction(p=0.04) even under induced strain (FIG. 3B). The same muscle sampleswere analyzed by confocal microscopy to detect and quantify dystrophinexpression at 90 days after hMDCC treatment. On average, 17% of thecells were positive for dystrophin expression.

Currently, there is no effective therapy to treat DMD, a lethal genetic,neuromuscular disorder affecting 1 in every 3500 newborn boys. Treatmentmodalities, such as growth-modulating agents, anti-inflammatory orsecond-messenger signal-modulating agents, and molecular devicesdesigned to skip mutations in the dystrophin gene have been attempted,however these approaches fail to halt or reverse disease progression. Bycomparison, the instant MDCC therapy represents a universal regenerativemedicine approach for local or systemic application in patientssuffering from DMD. Further, compared to other cell-based therapies, theunique features of MDCC, created through ex vivo fusion, are thecombination and synergistic effects of the complementing characteristicsof cells of myogenic and mesenchymal origin, such as high proliferationrate, capability for myogenic conversion, and secretion ofimmunomodulatory cytokines and growth factors facilitating muscleregeneration. Moreover, the preparation of MDCC does not require geneticmanipulations or introduction of viral vectors to the cells, thus makingit a safer therapy. In addition, since MDCC therapy is notgene/mutation-specific, it can be tailored and applied to patientssuffering from other types of muscular dystrophies including, e.g.,Becker dystrophy.

Example 2: Ex Vivo Preparation of Mouse Duchene Muscular DystrophyChimeric Cells (mDMDCC)

Healthy snj and mdx (dystrophin-deficient) primary myoblast cultures aswell as mdx MSC cultures were established and expanded in vitro. Threemurine myoblast/MSC and myoblast/myoblast fusions were performed withpolyethylene-glycol technique. Dystrophin expression, as well asmyogenic differentiation capacity, was confirmed before and afterfusion.

In in vivo studies (Table 6), efficacy of murine DMDCC engraftment,survival and restoration of dystrophin expression and motor functionswere tested 30 days following local delivery of mDMDCC to thegastrocnemius muscle. Of the five groups tested, mDMDCC composed ofmyoblast/myoblast and MSC/myoblast origin (dose 0.5×10⁶) were deliveredthrough multiple intramuscular injections following a standardizedtemplate, to the left gastrocnemius muscle of mdx mice. Control groupsincluded treatment with vehicle, treatment with not unfused myoblast andtreatment with mixed MSC and myoblast. Outcome measurements included invivo muscle function, dystrophin expression in treated muscles as wellas mDMDCC engraftment, which were assessed after 4 weeks. Aftermyoblast/myoblast mDMDCC delivery, dystrophin expressing cellsconstituted 37% of total nucleated cells on immunofluorescence analysisby confocal microscope. Myoblast/myoblast mDMDCC-treated muscle showeddystrophin-positive and dystrophin-negative areas. Dystrophin-positivecells were characterized by dystrophin expression on the membrane(normal pattern) as well as cytoplasmic expression.

TABLE 6 Time-point No. of Treatment (days) Animals Outcomes Myoblast/MSCMDCC 90 4 Muscle function and structure, DE Myoblast/Myoblast 90 4 MDCCengraftment, MDCC muscle function and structure, DE Myoblasts without 904 MDCC engraftment, fusion muscle function and structure, DE Myoblastand MSC 90 4 MDCC engraftment, without fusion muscle function andstructure, DE Vehicle 90 4 MDCC engraftment, muscle function andstructure, DE

Interestingly, mDMDCC-treated muscles showed decreased muscle weight ascompared to contralateral untreated muscles. The lower weight of thetreated muscle can be explained by decreased DMD-related hypertrophy andfibrosis. Despite the reduced muscle weight, muscle force measurements,evoked with electric stimulation in situ, resulted in increased valuesof muscle strength in vivo (FIGS. 4A and 4B) as well as ex vivo (FIGS.5A and 5B).

In vivo motor function evaluated by grip strength and wire hanging testsalso confirmed the efficacy of mDMDCC with increased grip strength andprolonged time to resist fatigue. Myoblast/Myoblast mDMDCC-treatedmuscle maintained higher strength values than muscles treated withmyoblast/MSC MDCC, which did not differ from control muscle values 30days after therapy delivery. Myoblast-derived mDMDCC treatment resultedin increased tolerance for fatigue, shown by longer hanging time on wirecompared to control animals.

What is claimed is:
 1. An isolated Muscular Dystrophy Chimeric Cell(MDCC) population comprising cells produced by ex vivo fusion andexpansion of (a) a first myoblast; and (b) a second myoblast,mesenchymal stem cell, or stromal cell, wherein at least one of thefirst myoblast, second myoblast, mesenchymal stem cell, or stromal cellis from a healthy donor and the other is a dystrophin deficient cellfrom subject suffering from a muscular dystrophy.
 2. The MDCC of claim1, wherein the first myoblast and second myoblast are from differentdonors.
 3. The isolated MDCC of claim 1, wherein the first myoblast,second myoblast, mesenchymal stem cell, or stromal cell is autologous orallogeneic.
 4. The isolated MDCC of claim 1, wherein the healthy donoris the subject's father.
 5. The isolated MDCC of claim 1, wherein themesenchymal stem cells are derived from bone marrow or adipose tissue.6. The isolated MDCC of claim 1, wherein said MDCC secretes one or moreimmunomodulatory cytokines and growth factors.
 7. The isolated MDCC ofclaim 6, wherein the one or more immunomodulatory cytokines and growthfactors comprise insulin-like growth factor 1, hepatocyte growth factorand myostatin.
 8. The isolated MDCC of claim 1, wherein said an ex vivofusion is between: a) a dystrophin deficient human myoblast from asubject suffering from a muscular dystrophy and a human myoblast from ahealthy donor; b) a dystrophin deficient human myoblast from a subjectsuffering from a muscular dystrophy and a human mesenchymal stem cellfrom a healthy donor; or c) a human myoblast from a healthy donor and adystrophin deficient human mesenchymal stem cell from a subjectsuffering from a muscular dystrophy; or d) a dystrophin deficient humanmyoblast from a subject suffering from a muscular dystrophy and a humanstromal cell from a healthy donor.
 9. A composition comprising theisolated MDCC of claim 1 and a pharmaceutically acceptable carrier. 10.A method of treating a muscular dystrophy comprising administering tothe subject in need of treatment an effective amount of the isolatedMDCC of claim 1 thereby treating the subject's muscular dystrophy. 11.The method of claim 10, wherein the muscular dystrophy is DuchenneMuscular Dystrophy.
 12. The method of claim 10, wherein said MDCC isadministered by intravenous injection, intra-bone injection orintramuscular injection.